Group A Streptococcus (GAS, S. pyogenes) causes a variety of human diseases ranging from mild pharyngitis and skin diseases to severe necrotizing fasciits, toxic shock syndrome, and autoimmune diseases. We have reported earlier that GAS upon interaction with host cells invokes intracellular signaling events and regulates protein phosphoryaltion in pharyngeal cells. The presence of the eukaryotic-type Ser/Thr kinase (ESTK) and protein phosphatase (ESTP) in prokaryotes and their functional importance in terms of regulatory roles in prokaryotic metabolic processes and signal transduction pathways have been recently recognized. However, their precise function is not known, since in most organisms, several genes encode for structurally similar proteins. GAS genome sequence analyses have also revealed the presence of a single gene each encoding for ESTK and ESTP, which are annotated as STK and pppL respectively. The information on the role of ESTK and/or ESTP in gram-positive bacterial pathogenesis is extremely limited. In particular, the role of STK and pppL in GAS pathogenesis is not known. In this proposal, we have identified, characterized and cloned STK and pppL specific genes, produced corresponding recombinant proteins and generated STK and pppL-specific polyclonal antibodies. Mutants strains, lacking STK or its C-terminal outer domains, exhibited remarkable changes in the expression of surface proteins and cell division profiles as revealed by scanning and transmission electron microscopy. With these results, we hypothesize that STK and pppL play a significant role in GAS pathogenesis. We propose several strategies to create and characterize mutant strains lacking the expression of kinase domain of STK, pppL, and both STK and pppL by genetic, biochemical and immunological analyses. In subsequent Specific Aims, we will test virulence of all the mutant strains using in vitro assays for GAS adherence to and invasion of pharyngeal cells and its phagocytosis by human neutrophils and confirm their virulence potential by specific antibody-mediated adherence inhibition and opsonophagocytosis assays. These in vitro findings will be validated with in vivo mouse intraperitoneal and nasopharyngeal infection models. The results obtained from this exploratory grant will allow to understand the roles of these novel signaling molecules in bacterial pathogenesis in general and GAS pathogenesis in particular.